\chapter{Recipies}

\section{Reagents}

\subsection{\acf{DMEM}}
\label{sec:dmem}
\acf{DMEM} (Gibco cat 41965-039).

\subsection{\acf{CS}}
\acf{CS} (Gibco cat  16010-084).

\subsection{Trypsin}
Trypsin (0.25\%)/EDTA (1 mM) solution in Hanks Balanced salt solution.  (Gibco 25200-056)

\subsection{\acf{PS}}
\acf{PS}. All media is supplemented with streptomycin sulfate (f.c. 0.1 mg/ml) and penicillin G (f.c.100 U/ml).  Use 100 x solution (10,000 units penicillin and 10 mg streptomycin per ml): Sigma P0781

\subsection{\acf{FBS}}
\acf{FBS}. (Gibco cat  10081-164). Again each lot must be tested. In both cases, get Gibco to match the characteristics of the current lot in the new one.

\subsection{\acf{PBS}}
\label{recp:pbs}

\acf{PBS}: Cambrex, BioWhittaker DPBS Cat No. BE17-512F

\subsection{HEPES}
\label{recp:hepes}

\acf{HEPES}

\subsection{EDTA}
\label{recp:edta}

\acf{EDTA}

\subsection{PMSF}
\label{recp:pmsf}

\acf{PMSF}


\subsection{Protease Inhibitors}
\label{recp:prot-inhib}
\ac{TODO} Complete, Mini, EDTA-free - Roche


\subsection{Insulin}
\label{recp:insulin}
Insulin: Novo Nordisk Human Actrapid (100 iu/ml)


\subsection{\acf{TNF}}
\label{recp:tnf-powder}

\ac{TNF} (Sigma cat T 7539). 



\section{Buffers}

\subsection{\acf{DMEM}/10\% \acf{CS}}
\label{recp:DMEM10CS}

\begin{table} [ht]
\caption[\acf{DMEM}/10\% \acf{CS}]{The recipie for acf{DMEM}/10\% \acf{CS}}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	\ac{DMEM} & 500 ml & Gibco, 41965-039) \\
    \ac{PS} & 5 ml & (Sigma, P0781) \\
    \ac{CS} & 56 ml & (Gibco cat 16010-084)	 \\
\hline
\end{tabular}
\end{center}
\end{table}

\subsection{Adipocyte medium}
\label{recp:adipopcyte-medium}

\begin{table} [ht]
\caption[Adipocyte medium]{The recipie for adipocyte medium}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	\ac{DMEM} & 500 ml & Gibco, 41965-039) \\
    \ac{PS} & 5 ml & (Sigma, P0781) \\
    \ac{FBS} & 56 ml & (Gibco, 10081-164) \\
\hline
\end{tabular}
\end{center}
\end{table}





\ac{PS}: All media is supplemented with streptomycin sulfate (f.c. 0.1 mg/ml) and penicillin G (f.c.100 U/ml).  Use 100 x solution (10,000 units penicillin and 10 mg streptomycin per ml): Sigma P0781  





\section{Differentiation medium}
\label{sec:diff-medium}

\begin{table} [ht]
\caption[Differentiation medium]{The recipie for differentiation medium}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Adipocyte medium (see section \ref{recp:adipopcyte-medium})  & 100 ml & - \\
    \ac{IBMX} & 0.5mM (add 194 $\mu$l of stock) & - \\
    dexamethasone & 0.25 $\mu$M (add 194 $\mu$l of the DEX/DMEM/FBS stock (see Section \ref{sec:dex})) NOTE THIS IS NOT THE CONCENTRATED DEXAMETHASONE & sigma, D1756	 \\
    Insulin (see reagent \ref{recp:insulin}) & 1$\mu$g/ml (add 22 $\mu$l of stock) & - \\
\hline
\end{tabular}
\end{center}
\end{table}

\subsection{DEX/DMEM/FBS}
\label{sec:dex}

Dexamethasone: (sigma, D1756; FW 392.5)
Stock Solution: 2.5 mM in EtOH (0.98 mg/ml). Dilute 1/20 with DMEM + 10 \% FBS immediately prior to use to yield a 500 x solution.
24.5 mg Dexamethasone dissolved in 25 ml 100 \% EtOH
Store at 4C for 6 months or less in a clear plastic tube.

\subsection{Methyl Isobutylxanthine solution}
Methyl Isobutylxanthine: (Sigma, I5879; FW  222.2)
500x solution: 55.6 mg IBMX dissolved in 1.0 ml 0.35 N KOH.
(Calculation for 0.35M KOH, Molecular weight KOH  = 56.11
0.35x56.11 = 19.63g/L
                   		       = 0.49g/25ml         )
Load into 3 ml syringe with large bore needle.
Filter through 0.22 micron filter (SS uniflow-4602330) into 12 ml plastic-tube.
Make this solution fresh the day of use.

\subsection{Insulin medium}
\label{sec:insulin-medium}

\begin{table} [ht]
\caption[Insulin medium]{The recipie for insulin medium}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Adipocyte medium (see Section \ref{recp:adipopcyte-medium}) & 100 ml & - \\
    Insulin & 1 $\mu$g/ml (add 22 $\mu$l of stock) & (Sigma, P0781) \\
    \ac{FBS} & 56 ml & (Gibco, 10081-164) \\
\hline
\end{tabular}
\end{center}
\end{table}

\subsection{Homogenisation buffer}
\label{sec:homog-buffer}

\begin{table} [ht]
\caption[Homogenisation buffer]{The recipie for homogenisation buffer (300 ml). Make up to 300 ml, pH 7.4 with NaOH }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Sucrose & 225 mM (MW = 342.3, 23.1 g / 300 ml) & Sigma - \\
    HEPES (section \ref{recp:hepes}) & 20 mM (MW = 238.3, 1.44 g / 300 ml) & Sigma -  \\
    EDTA (section \ref{recp:edta}) & 1 mM (MW = 292.25, 0.087 g / 300 ml) & Sigma -  \\
\hline
\end{tabular}
\end{center}
\end{table}



\subsection{Complete Homogenisation Buffer}
\label{sec:complete-homog-buffer}

Make up Complete Homogenisation buffer in yellow eppendorfs (low protein binding affinity)

\begin{table} [ht]
\caption[Complete Homogenisation buffer]{The recipie for Complete homogenisation buffer. }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Homogenisation buffer (section \ref{sec:homog-buffer}) & 1 ml & - \\
    Protease Inhibitor Solution (section \ref{recp:prot-inhib-solution}) & 150 $\mu$l & -  \\
    PMSF (section \ref{recp:pmsf}) & 4$\mu$l (250 mM stock) & Sigma   \\
C12 E9 & 28$\mu$l (0.5 \%) & Sigma   \\	
\hline
\end{tabular}
\end{center}
\end{table}


\subsection{Protease Inhibitor Solution}
\label{recp:prot-inhib-solution}

Dissolve one tablet of Complete, Mini, EDTA-free (see section \ref{recp:prot-inhib}) in 1.5 ml redistilled H20 (7xconc)


\section{\ac{TNF} Solution}
\label{recp:tnf-soln}

\begin{enumerate}
 \item Add 1ml of Adipocyte medium (See section \ref{recp:adipopcyte-medium}) to \ac{TNF} powder (See section \ref{recp:tnf-powder}) in vial (10$\mu$g)
 \item Add further 666$\mu$l of Adipocyte medium (See section \ref{recp:adipopcyte-medium}). Final volume 1.667 $\mu$l
 \item Master stock at 6$\mu$g/ml
 \item Do a 1:10 dilution and add 50$\mu$l to each plate.
\end{enumerate}

\section{\ac{TNF}/Adipocyte medium}
\label{recp:tnf-adipo-medium}

Make up \ac{TNF} solution first (See Section \ref{recp:tnf-soln}) 10 ml of Adipocyte medium represents 1 plate, therefore add 50$\mu$l of \ac{TNF} solution (section \ref{recp:tnf-soln}) to 10 ml of Adipocyte medium.

\begin{table} [ht]
\caption[\ac{TNF}/Adipocyte medium]{The recipie for \ac{TNF}/Adipocyte medium. }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Adipocyte Medium (section \ref{recp:adipopcyte-medium}) & 10 ml & - \\
    \ac{TNF} powder (section \ref{recp:tnf-powder}) & 30 ng  & Sigma cat T 7539  \\
\hline
\end{tabular}
\end{center}
\end{table}


\section{Adipocyte Medium/10nM Insulin}
\label{recp:adipo-insulin}

\begin{table} [ht]
\caption[Adipocyte Medium/10nM Insulin]{The recipie for Adipocyte Medium/10nM Insulin. Add 17 $\mu$l of resulting solution per plate (10 ml of adipocyte medium) for 10nM solution. }
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Adipocyte Medium (section \ref{recp:adipopcyte-medium}) & 500 ml & - \\
    Insulin (section \ref{recp:insulin}) & 5 $\mu$l  & -  \\
\hline
\end{tabular}
\end{center}
\end{table}

\section{HES} 
\label{recp:hes}

	

\begin{table} [ht]
\caption[HES Buffer]{The recipie for \ac{HES} Buffer. Add the following to 500ml water and adjust the pH to 7.4 and store at 4 $^\circ$C}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	Sucrose & 255 mM (43.6 g) & - \\
    \ac{HEPES} & 20 mM (2.38g)  & -  \\
	\ac{EDTA} & 1 mM (0.186g)  & -  \\
\hline
\end{tabular}
\end{center}
\end{table}

\section{1.12M sucrose in HES}
\label{recp:sucrose-hes}

Add 3.834g sucrose to a small volume of HES buffer (Section \ref{recp:hes}) (sufficient to allow dissolution) then make the volume up to 10 ml with HES.